This disclosure relates generally to sampling natriuretic and other peptides in biological fluids, such as blood, urine and the like. More particularly, it relates to preserving a profile of proteins and peptides of interest in the sampled biological fluid for analysis as continued proteolysis or modification of such peptides and proteins may make subsequent analysis suspect. This disclosure is also useful for monitoring performance of BNP test procedures and biochemical markers used for diagnosis and staging of patients with Congestive Heart Failure (“CHF”).
Heart Failure (“HF”) compromises ventricular systolic or diastolic function, or both due to cardiac insufficiency. This reflects the inability of the heart to pump sufficient oxygen-rich blood to accommodate the body's needs. CHF further includes the accumulation of fluids in the lungs and breathlessness (dyspnea). The heart responds to this perturbation of fluid homeostasis by secreting natriuretic peptides, which assist in combating the accumulation of fluids in the lungs and other effects of CHF and HF.
Natriuretic peptides are a class of hormones that regulate blood pressure, electrolyte balance, and fluid volume. Natriuretic peptides secondary structure includes a loop formed by an internal disulfide bond between two cysteine residues as shown for Atrial natriuretic peptide (“ANP”) and B-type natriuretic peptide (originally referred to as ‘Brain Natriuretic Peptide,’ (“BNP”)) in FIG. 1. Other natriuretic peptides of interest are C-type natriuretic peptide (“CNP”) and Dendroaspis natriuretic peptide (“DNP”). The latter was originally isolated from mamba snake venom. See, e.g., US Patent Publication No. 20020082219. CNP is primarily secreted by brain substructures and by the endothelium. It is primarily found in the brain and cerebrospinal fluid with little if any present in the heart.
The structure, function and processing of natriuretic peptides is conserved across species. All known natriuretic peptides seem to have an internal loop formed by a disulfide bond. DNP exhibits further similarity to ANP, BNP and CNP and antibodies to DNP tag some Human epitope(s). DNP has an action similar to that of ANP and BNP, see, e.g., Singh et al. in Circulation Res. (DOI: 10.1161/01.RES0000232322.06633.d3 published online on Jun. 15, 2006), and seemingly has a longer half-life compared to ANP and BNP. See, e.g., Lisy et al. (2001) Hypertension 37, pp. 1089-1094. Indeed, putative homologs of human natriuretic peptides have been isolated in species as distant as fishes including lampreys and hagfishes. See, e.g., Kawakoshi et al. (2006) General and Comparative Endocrinology 148, pp. 41-47. These distantly related peptides exhibit conserved processing and cleavage sites and the loop structure illustrated in FIG. 1.
ANP is a 28-amino acid hormone that originates from the atria of the heart. Within the myocyte, ANP is synthesized as a prepro-ANP (a 151-amino acid peptide), which is cleaved to yield pro-ANP (a 126-amino acid peptide). Pro-ANP is further processed by protease Corin, see, e.g., Wu et al. (2005) Biochimica and Biophysica Acta. 1751:1, pp. 82-94. Pro-ANP can also be processed by protease Furin, at least in vitro, and serum proteases, such as kallikrein and thrombin to yield the 28-amino acid active ANP peptide. See, e.g., Gibson et al. (1987) Endocrinology 120, pp. 764-772. The 28-amino acid ANP peptide is further degraded by a neutral endo-peptidase (EC 3.4.24.11). See, e.g., Wu et al. supra.
BNP is a 32-amino acid peptide secreted by myocytes and fibroblasts in the ventricles in response to increased wall stretch and volume overload. Within the myocyte, BNP is synthesized as prepro BNP (a 134-amino acid peptide), which is cleaved to yield the secreted proBNP (a 108-amino acid peptide). ProBNP is further processed to yield the 32-amino acid active BNP peptide by yet to be definitively identified proteases. In vitro, proteases like Corin, see, e.g., Wu et al. supra, and Furin, see, e.g., Sawada et al. (1997) J. Biol. Chem. 272:33 pp 20545-20554 can process prepro BNP. The 32-amino acid BNP peptide can be further cleaved by endo-peptidases like Dipeptidyl-Peptidase IV to yield a 30-amino-acid active natriuretic peptide, which has been observed in vitro. In vivo, the 30-amino acid peptide is subjected to proteolysis to generate several peptides. Multiple peptide fragments of pro-BNP have been detected in plasma. See, e.g., Shimizu et al. in Clinica Acta. (2002) 316, pp. 129-135. The 32-amino acid BNP peptide may be also generated by proteases in the circulating blood fluids. See, e.g., Hunt et al. (1997) Peptides 18, pp. 1475-1481.
In general, plasma levels of natriuretic peptides reflect a balance between secretion of the propeptide, proteolytic processing and their clearance. BNP is inactivated by proteolysis in addition to receptor-mediated clearance and filtration by kidneys.
Plasma BNP concentration is one of the most sensitive and specific indicator of congestive heart failure. Plasma concentration of BNP related peptides is sharply elevated in patients with CHF. As a result, BNP related peptides are often evaluated in patients arriving at the emergency room with dyspnea. Presently, some assays for the amino portion of pro-BNP detect both pro-BNP and the cleaved N-terminal part of pro-BNP, Nt-proBNP. As a result, the assay fails to accurately estimate pro-BNP levels. Similarly, considerations apply to other assays.
Nt-proBNP and BNP levels are reliable indicators or markers of clinical severity and left ventricular ejection fraction as well as morbidity and mortality. In recent years, Nt-proBNP and BNP have been used to diagnose and classify CHF severity. According to the CHF classification adopted by the New York Heart Association (“NYHA”), the mean concentrations of BNP progressively increase from stage I to IV. For instance, mean BNP concentrations of 71 pg/ml, 204 pg/ml, 349 pg/ml, and 1022 pg/ml corresponded to CHF stages I through IV respectively. Stage IV of CHF represents the highest severity of cardiac disease resulting in inability to carry on any physical activity without discomfort. A patient in this stage of the disease may have symptoms of heart disease or the coronary syndrome even at rest with increasing discomfort if any physical activity is undertaken.
Much of the detectable BNP in circulation in patients suffering from CHF seems to be in the form of relatively inactive precursors or fragments of BNP. Many additional undetectable (by assays in use presently) fragments of BNP may well be circulating with unknown biological effects. See, e.g., Heublein et al. (2007) Hypertension 49, pp. 1114-1119. Sampling errors due to continued processing of BNP related peptide fragments add to the uncertainty in evaluating BNP production by a subject.
Entire BNP, BNP1-32, reportedly is a substrate for endopeptidase Dipeptidyl-Peptidase IV (DPP IV), see, e.g., Brandt et al. (2006) Clinical Chemistry 52, pp. 82-87, which shortens it by removing two N-terminal residues to generate BNP3-32, which is also biologically active. BNP3-32 is further degraded by other proteases, see, e.g., Pankow et al. in Circulation Res. of Sep. 6, 2007 online publication DOI:10.1161/CIRCRESAHA.107.153585 to generate additional peptides. Proteases like thrombin, plasmin and the like are also capable of cleaving pro-BNP at least in vitro. See, e.g., Hunt et al. Supra. Such proteolysis is expected to add to sampling errors due to continued proteolysis of sampled BNP. BNP also induces Matrix Metalloproteinases. See, e.g., Tsuruda et al. (2002) Circulation Res. 91, pp. 1127. Some metalloproteinases are known to further proteolyze BNP, see, e.g., Pankow supra. Thus, a large number of proteases are a potential source of sampling errors.
As a result, sampling the blood or another biological fluid from a patient typically provides an inaccurate representation of BNP related peptides, information that is important for diagnostic applications and other uses. See, e.g., Daniel L. Dries (2007) Hypertension 49, pp. 971-973. This lack of clarity is due to the possibility that BNP related peptides continue to be subject to proteolysis after sampling, including proteolysis by proteinases induced by BNP or the act of sampling itself.
There have been many attempts to employ sampling and processing methods to reduce or eliminate the artifacts introduced by such ongoing proteolysis. Many serine proteases are known to act on BNP, its proteolytic products or its precursors. For non-fluid tissues, boiling in water is assumed to inactivate proteases. See, e.g., Hunt et al. in Peptides (1997) 18, pp. 1475-1481. Investigators purifying BNP from tissue have reported the use of serine proteases inhibitors like aprotinin or a combination of aprotinin and benzamidine for preventing BNP proteolysis. See, e.g., Tsuji et al. in Clin. Chem. (1994) 40, pp. 672-3. Among the many suggestions for controlling induction of proteases by the act of sampling is the use of plastic tubes. Also available are blood collection tubes containing protease inhibitors. An example is BD P100 v1.1 blood collection vacuum tubes (Becton, Dickinson, and Company catalog no. 8013142). Protease inhibitors like PPACK I, PPACK II and Protease inhibitor cocktail set III from Calbiochem provide broad spectrum protease inhibition. However, these inhibitors are not entirely suitable for applications such as sampling natriuretic peptides and their fragments or precursors due to both technical limitations and their cost (see infra). When sampling blood, protease inhibitors like AEBSF and Benzamidine have been used to arrest proteolysis at relatively low concentrations—in part to avoid covalent modification of the sampled proteins.
Many insufficiently effective approaches have been adopted to reduce proteolysis of BNP and other peptides. These methods include use of plastic tubes or adding EDTA to the collected samples or broad-spectrum protease inhibitors ROCHE™ supplies AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a water-soluble serine protease inhibitor with a molecular weight of 239.5 Da under the trade name PEFABLOC SC™. AEBSF inhibits proteases like chymotrypsin, kallikrein, plasmin, thrombin, and trypsin. Typical working concentrations are in the range of at 0.1-1.0 mM with stock solutions at 100 mM.
As to AEBSF, ROCHE™ provides notice that at high concentrations it forms covalent adducts with proteins and peptides. Thus, such inhibitors are used at about the recommended concentration range, see, e.g., the disclosed use of AEBSF at 0.125 mM in US Patent Publication No. 2006/0183681 (discussed infra). However, ROCHE™ also sells a proprietary composition together with the inhibitor in a package, e.g., PEFABLOC SCPLUS™ (or PEFABLOC SC PLUS™). It is unknown if formation of adducts is adequately prevented at concentrations of interest for sampling blood and plasma like fluids. Further, as discussed above, proteases in addition to serine proteases are responsible for and capable of degrading BNP and related peptides in plasma, which proteases are not effectively inhibited by AEBSF.
An illustrative example, US Patent Publication No. 2006/0183681 (“the '681 publication”) describes well-known protease inhibitors to prevent further hydrolysis of BNP. The '681 publication teaches preparing a serum based standard, see, e.g., paragraph 42 of the '681 publication, by pooling sampled blood plasma, defibrinating it followed by delipidizing it and then adjusting the total protein concentration followed by the addition of protease inhibitors benzamidine and AEBSF to a final concentration of 9.5 mM and 0.125 mM respectively and then spiking it with a predetermined amount of BNP. This serum-based preparation is stable for several days at −20° C. The publication does not describe the process by which plasma is obtained or the effect of the long time taken to process the sampled plasma on the level of endogenous BNP and peptides related to it. Indeed, sampling biological fluids is not addressed nor are problems peculiar to such sampling identified or any solutions suggested.
US Patent Publication 2004/0067889 (“the '889 Publication”) discloses compositions for preserving BNP in sampled biological fluids. The '889 Publication discloses that protease inhibitors PPACK and PPRACK are most effective in arresting proteolysis of BNP in sampled blood or plasma. Further, these inhibitors are effective alone or in combination with AEBSF, leupeptin and antipain as well as benzamidine. The '889 Publication posits that structural similarity between protease inhibitors and regions of BNP is responsible for the effectiveness of the protease inhibitors in stabilizing BNP.
The '889 Publication does not disclose effective and efficient compositions for sampling a biological fluid and preserving a peptide profile therein based on synergy between two or more constituents of the compositions. Further, the '889 Publication does not disclose efficient and effective protease inhibitor compositions that do not exhibit artifacts due to formation of adducts with the sampled peptide or protein. Indeed, PPACK and PPACK II are expensive protease inhibitors, indeed significantly more expensive than AEBSF. Therefore, an efficient method for preventing proteolysis after sampling is not disclosed to one having ordinary skill in the art.
A number of point-of-care diagnostic tests for BNP are available. ABBOTT AxSYM™, BAYER ADVIA CENTAUR™, and BIOSITE TRIAGE™ BNP assays are some of the most widely used quantitative test methods for determination of BNP. The ABBOTT AxSYM™ assay utilizes the Microparticle Enzyme Immunoassay (MEIA) technology, which uses microparticles coated with anti-BNP monoclonal antibodies that bind to human BNP antigen. These antigen-antibody complexes on the microparticles bind a monoclonal anti-BNP alkaline phosphatase conjugate capable of yielding a fluorescent product. The fluorescent intensity is used to determine BNP levels. The BIOSITE TRIAGE™ BNP assay is an immunofluorometric assay. In this assay, a rabbit recombinant polyclonal antibody is bound to the fluorescent label, and a murine monoclonal antibody against the disulfide bond-mediated ring structure of BNP is bound to the solid phase. In this assay plasma is treated with fluorescent antibody conjugates and complexes of BNP and the fluorescent antibody conjugate are captured on a detection lane. The concentration of BNP in the specimen is proportional to the fluorescence from bound complexes. The BAYER ADVIA CENTAUR™ assay is a two-site sandwich immunoassay with an acridinium ester labeled monoclonal mouse anti-human BNP (specific to the ring structure on BNP) as the first antibody and a biotinylated monoclonal mouse anti-human antibody (specific to the C-terminal portion of BNP) as the second antibody (solid phase). The complex is further coupled to streptavidin magnetic particles. The lower limits of detection for the ABBOTT AxSYM™, BIOSITE TRIAGE™, and BAYER ADVIA CENTAUR™ BNP assays are 15, 5, and 2 pg/mL, respectively.
BNP and similar peptides exhibit poor stability in serum or plasma. BNP is cleared from circulation by specific cellular receptors and proteases including endopeptidases. A reason for the poor stability of BNP, in addition to proteolysis by multiple natural proteases in plasma or serum, is due to excretion by the kidneys and clearance by uptake through Natriuretic Peptide Receptor C (NPR-C), which in kidneys is primarily distributed in the podocyte region. NPR-C potentially provides a mechanism to down regulate BNP levels. As a result, the half-life (t1/2) of BNP in vivo is of the order of approximately 20 minutes or so.
Notwithstanding the above difficulties, plasma BNP concentration is a preferred marker for diagnosis and prognosis of cardiac function and acute myocardial infarction. Plasma concentrations of BNP increase with a decline in heart function. Potentially BNP can serve as the preferred biochemical marker for pre-screening patients for further cardiac investigations and/or treatment. However, the limited stability of sampled BNP makes this difficult. In vitro, BNP is rapidly proteolyzed, for example, within 24 h of separation of plasma from whole blood. See, e.g., Belenky et al. in Clinica Chimica Acta (2004) 340, 163-172. Progressive degradation during refrigerated storage makes accurate measurement of BNP challenging.
Thus, currently used quantitative tests for BNP likely include avoidable errors due to continued proteolysis of proBNP and BNP fragments after sampling. Such errors may result in either overestimation or underestimation of circulating BNP related peptide species.
The sampling technique itself becomes important for determining the level of the peptide of interest in general even in case of peptides other than natriuretic peptides.
Therefore, there exists a need for a sampling method, device and composition to allow accurate sampling of BNP and other peptides in biological fluids for subsequent assays.